This invention relates to a de-staining composition, in particular a composition for de-staining stained substrates such as electrophoresis gels.
One of the most widespread techniques in modern molecular biology, biochemistry, clinical analysis etc. is electrophoresis as performed on polyacrylamide or other gels. In this procedure a mixture of substances, typically proteins or peptides, in a biological sample is separated by virtue of the differential mobility of the individual components in an electrical field. For this method to be useful it is necessary to reveal the usually colourless separated bands of material.
Typically in such gel-electrophoresis procedures, the development of the bands corresponding to the individual proteins, peptides or other biological compounds under investigation requires a step in which these compounds are stained with dyes, typically lasting 30-60 min. This is generally then followed by a separate and lengthy de-staining step for example in a methanol-acetic acid mixture to remove excess dyes from the gel thus clearing the background so that the stained bands may be seen. Usually the de-staining step takes a much longer time than the staining step.
Used de-staining solutions are typically recycled by passing through a column of activated charcoal or ion exchange resin, which binds the excess staining dyes. More often, in research laboratories, simple funnels, filled with activated charcoal, are used instead of columns. This requires use of a fume hood to remove the harmful vapours of the solvent, typically methanol and acetic acid.
U.S. Pat. No. 4,357,174 describes a de-staining apparatus where a stain absorbing material (granulated charcoal) is located in a housing with apertures and retained therein by a porous material disposed around the internal walls of the housing. However, the efficiency of this apparatus is limited by the surface area of the apertures restricting the amount of de-staining fluid allowed to flow into and out of the housing.
Other proposals have been made for simplifying the various de-staining techniques such as diffusion, in-flow filtration and use of electrical fields. However, these proposals involve either high cost apparatus requiring highly qualified personnel and sophisticated techniques for production and/or suffer from relatively low efficiency such as in U.S. Pat. No. 4,357,174 as described above.
It is an object of the present invention to address these problems by providing inter alia a cheap, efficient and easy to use de-staining composition.
The invention therefore provides a de-staining composition for de-staining a stained substrate, which composition comprises an absorbent for the stain incorporated in a semi-solid matrix.
Advantages of the compositions according to the invention reside inter alia in their adaptability to different uses, ease of handling and convenience.
The semi-solid matrix can be made from any material which will permit incorporation of the absorbent and remain semi-solid once the absorbent is incorporated and in use.
Preferably the semi-solid matrix is a gel.
An especially preferred gel is a polyacrylamide or agar gel.
When an agar gel is employed it is preferred that the agar gel is an agarose gel.
Suitable absorbents for the stain include charcoals (activated or otherwise) or ion exchange resins such as styrene-divinyl benzene copolymers having sulfonic acid or quaternary amine groups.
Preferred absorbents for the stain are activated charcoal or a mixture of ion exchange resins.
The absorbent material will typically be incorporated into the semi-solid matrix prior to or during formation of the latter by mixing. Thus, by way of example, in the case of an agar gel the absorbent is mixed into a heated suspension or solution of the agar which is then allowed to set in appropriately sized moulds. In the case of polyacrylamide gels the absorbent may be mixed into the acrylamide prior to polymerisation.
The concentration of the gel itself and of the absorbent incorporated therein will be chosen in accordance with the particular use for which the composition is intended and will be within the competency of the skilled person.
For example, in the case of gels, the concentration will preferably be in the range of 0.5 to 10% w/v, especially 1 to 5% w/v.
In these gels, the concentration of absorbent will preferably be in the range of 0.5 to 10% w/v especially 2 to 5% w/v.
The size and shape of the semi-solid matrix once formed will be chosen in accordance with the type of substrates it is desired to de-stain, the apparatus in which de-staining is to take place and the capacity of the composition in terms of its ability to absorb stain.
Preferably, the semi-solid matrix with the absorbent will be capable of being used a number of times before discarding or regenerating as desired.
According to a further aspect of the invention there is provided a method for de-staining a stained substrate, which method comprises bringing the substrate to be de-stained into contact with a de-staining composition as hereinbefore defined under conditions which will bring about release of the stain from the stained substrate.
Preferably, the de-staining composition and the substrate to be de-stained are brought into contact with a de-staining solution comprising a solvent or mixture of solvents capable of bringing about release of the stain from the stained substrate.
This method is of particular value when the substrate to be de-stained is a developed electrophoresis gel.
The solvent or mixture of solvents employed as the de-staining solution in the method according to the invention will be chosen in accordance with the nature of the stain it is desired to extract and absorb and the nature of the substrate which is to be de-stained. This choice will be within the capability of the skilled person in this field.
Typically the de-staining solution is or comprises an organic solvent.
De-staining solutions are preferred wherein the solvent is or comprises an alcohol. A preferred such solvent is an aqueous alcohol. Examples of alcohols include methanol, ethanol and butanol.
Other solvents may optionally be added to the de-staining solution.
Thus, optionally, the de-staining solution additionally comprises an organic acid. An example of such an organic acid is acetic acid.
A particularly preferred de-staining solution comprises aqueous methanol and acetic acid.
In carrying out the method in accordance with the invention the substrate to be de-stained and the composition according to the invention are brought together in such a way as to allow de-staining to take place. This can be achieved for example by placing the substrate to be de-stained and the composition according to the invention together with a de-staining solution capable of bringing about release of the stain in question in a suitable receptacle.
In a further aspect of the invention there is provided a method for carrying out staining and de-staining in a single operation. Thus, by using a staining solution of suitable composition, the entity to be stained will take up sufficient stain to achieve the requisite staining while simultaneously the de-staining composition will remove any excess stain so that the staining and de-staining steps need not be separated.
Thus, in one embodiment of the invention the de-staining composition is effective to remove any excess stain from a substrate, such that staining and de-staining can be carried out in a single operation.
Preferably, the de-staining composition is pre-equilibrated with the de-staining solution.
Further, preferably, the staining compound is in the form of a solution of said de-staining solution at a concentration in the range 0.01-0.1% w/v.
In this embodiment of the invention, the concentration of the absorbent for the stain entrapped in the semi-solid matrix has to be sufficient to capture all excess stain from the staining solution without bleaching the bands of the entity to be stained. As hereinafter demonstrated, we have established the necessary optimal conditions to achieve satisfactory results.
This aspect of the invention can be considered as a staining method which obviates the need for a separate de-staining step because the absorbent for the stain incorporated in the semi-solid matrix allows for the removal of excess stain in a single operation.
This aspect of the invention offers convenience to the scientist who, for example, runs an electrophoresis gel at the end of a day, is unable to wait for the gel to stain but nevertheless wishes to see the results first thing the next day.
Experimentally, it has been shown that if one uses solutions with dye concentrations of 0.01-0.1% w/v and methanol (10-30% v/v) and acetic acid (5-15% v/v), it is possible to get a clear background on a gel with sharp and well-stained protein bands over approximately 3-20 h depending on the composition of the solutions used.
References hereinafter to de-staining should also be read as embracing simultaneous staining and de-staining in accordance with the invention.
The stain as used herein is typically a dye, more especially a dye for proteins selected from Coomassie, Eosin, Procion, Cibacron, Fast and other dyes.
Examples of Coomassie dyes are:
Coomassie(copyright) Brilliant Blue R-250 (Brilliant Blue R);
Coomassie(copyright) Brilliant Blue G-250 (Brilliant Blue G);
Coomassie(copyright) Brilliant Cresyl Blue (Brilliant Blue C);
Coomassie(copyright) Brilliant Green; and
Coomassie(copyright) Violet R-200.
Examples of Eosin dyes are:
Eosin Yellowish (Eosin Y);
Eosin Scarlet, and others.
Examples of Procion(copyright) dyes are Procion(copyright) Blue MX-R and others.
Examples of Cibacron(copyright) dyes are Cibacron|(copyright) Blue F3G-A and others.
Examples of Fast dyes are:
Fast Green FCF (Food Green 3);
Fast Blue B Salt and others
Examples of other dyes include:
Naphthol Blue Black (Amino Black 10B);
Alcian Blue 8GX (Ingrain Blue 1);
Drimarene Brilliant Blue K-BL and others.
Probably the most commonly used dye is Coomassie(copyright) (Brilliant Blue R-250 (Brilliant Blue R) and the invention will hereinafter be illustrated with respect to this dye.
According to a further aspect the invention there is provided an apparatus for de-staining a stained substrate, which apparatus comprises a receptacle containing a de-staining composition as hereinbefore defined and which is capable of receiving and containing both the substrate to be de-stained and a de-staining solution comprising a solvent or mixture of solvents capable of bringing about release of the stain from the stained substrate.
Preferably, the semi-solid matrix forms a layer covering the base of the inside of the receptacle.
Alternatively, the semi-solid matrix is in the form of a block which will float in the de-staining solution when added to the receptacle.
The size and shape of the receptacle will be chosen in accordance with the substrate to be de-stained. For example when the substrate to be de-stained is an electrophoresis gel the receptacle will be in the form of a dish or tray.
The receptacle is made from a material which is inert to the de-staining solution. Suitable materials include glass and plastics materials such as methylmethacrylate.
Preferably, the receptacle is made from a transparent material. This allows a user to follow the de-staining process without having to open the apparatus.
Optionally, the receptacle bears a lid. This helps to reduce evaporation of the de-staining solution.
Preferably, the lid is made of a transparent material.
When the apparatus is of the base-covered type, all or part of the bottom of the receptacle will be provided with a white background to make detection of the gel background during the de-staining process easier.
The contents of the receptacle when in use, namely de-staining solution, substrate and de-staining composition can be agitated in conventional manner by direct stirring or by shaking and/or rocking on a conventional apparatus of the type used in electrophoresis.
In one embodiment the de-staining composition is effective to remove any excess stain from a substrate, such that staining and de-staining can be carried out in a single operation.
Preferably, the staining composition is pre-equilibrated with the de-staining solution.
Further, preferably, the staining compound is in the form of a solution of said de-staining solution at a concentration in the range 0.01-0.1% w/v.